Staining and confocal microscopy

To stain group-III auditory neurons, recording electrodes were filled with neurobiotin (1% m/v, SP-1120, Vector Laboratories) dissolved in intracellular saline. To aid diffusion of neurobiotin into the neurons a positive current of ∼200 pA was injected for ∼30 min. Directly after staining, Müller's organs were fixed overnight at 5°C in 4% paraformaldehyde (P6148, Sigma-Aldrich) dissolved in PBS. Müller's organs were then washed three times in PBS then gently shaken at room temperature for 20 min in a mixture of collagenase (0.5 mg/ml) and hyaluronidase (0.5 mg/ml; C5138 and H2126, Sigma-Aldrich). They were washed three times in PBS (3 × 10 min) then gently shaken at room temperature in 0.2% m/v Triton X-100 dissolved in PBS (2 × 60 min). Müller's organs were then gently shaken in 20 μg/ml Dylight 488 streptavidin (SA-5488, Vector Laboratories) and 0.05 mg/ml DAPI (D9542, Sigma-Aldrich) in PBS overnight at 5°C. During this time the fluorescent streptavidin binds very tightly to the fixed neurobiotin to specifically stain the recorded neurons. After this overnight incubation the Müller's organs were washed three times in PBS (3 × 10 min), dehydrated in an ethanol series and cleared in methyl salicylate (M6752, Sigma-Aldrich).

Fluorescence images (pixel size 0.31 μm², z stacks of 0.31 μm) were captured with a confocal microscope (FV1000 CLSM, Olympus) equipped with Plan-UPlanSApo 10× (0.4 numerical aperture) and 20× (0.75 numerical aperture) lenses. Fluorescence emission of Dylight 488 was collected through a 505–530 nm bandpass filter. Confocal images were adjusted for contrast and brightness, overlaid and stacked in ImageJ (v1.51, National Institutes of Health). The ImageJ plugin Simpler Neurite Tracer was used to determine the distance from the center of the soma to the dendrite dilation (see Fig. 3C).

Intracellular whole-cell patch-clamp recordings of auditory neurons, their spontaneous and tone-evoked transduction ion channel activity and neuron morphology. All spontaneous and tone-evoked transduction currents occur in the auditory neurons' cilium highlighted by a red circle on the neuron schematic on the left. A, Schematic showing experimental setup for intracellular patch-clamp recordings from Müller's organ housed on the internal surface of the tympanum perfused with saline. The outside of the tympanum is acoustically driven by airborne sound by a speaker. B, Double staining of the nuclei of cells of Müller's organ (magenta; DAPI) and all auditory neurons with whole nerve backfill using neurobiotin/Dylight 488 streptavidin (green). Group-III auditory neurons are highlighted by a white dotted circle. C, A neurobiotin-streptavidin staining of two group-III auditory neurons in situ reveals the dendrite dilation and apical cilium. D, Sound stimulation, voltage-clamp and recording protocol used to maximize the transduction current during intracellular patch-clamp recordings. A 3 kHz tone at 110 dB SPL (top trace) was used to stimulate group-III auditory neurons at their most sensitive frequency. The neurons were voltage-clamped to −100 mV (gray trace) to increase the electrochemical driving force. The extracellular saline contained 90 nm TTX and intracellular pipette solution contained 20 mm TEA to block sodium and potassium conductances and the spikes they facilitate. Discrete depolarizations (arrows) and the tone-evoked transduction currents are reduced in noise-exposed auditory neurons (red) compared with control (black). E, The amplitude of discrete depolarizations is reduced for auditory neurons from noise-exposed ears. The maximum six discrete depolarizations were averaged for each locust during the −100 mV voltage clamp. F, Example showing calculation of the latency of the tone-evoked transduction current. G, Latency of the transduction current was delayed for noise-exposed auditory neurons at 110 dB SPL and (H) across sound intensities. I, The transduction current increased with louder sound amplitudes, which was well fitted with a Boltzmann equation (solid lines). Mean is plotted as circles, positive SD is plotted as shaded area, transduction current amplitude from individual auditory neurons are plotted as thin shaded lines (Control: n = 12, N = 6; Noise-exposed: n = 12, N = 7). J, The maximal transduction current was significantly reduced at the maximal sound intensity of 110 dB SPL.